(1) Field of the Invention
The present invention relates to a method for evaluating atherosclerotic risk by means of the detection of levels of PHB in blood serum in humans. In particular the present invention relates to a method which utilizes an antibody for such detection.
(2) Prior Art
Atherosclerotic cardiovascular disease (CVD) is a leading cause of death in industrialized countries. Though it is current opinion that a variety of initiating agents and multiple interactive mechanisms contribute to the formation of atheromatous plaques, the critical role of lipids in the process is not disputed (Steinberg, P., In "Hypercholesterolemia and Atherosclerosis. Pathogenesis and Prevention", Churchill Livingstone, New York, pp 5-25 (1987)). Apparently, the focal deposition of lipids transported into the vessel wall by lipoproteins plays an important role in the initiation of an atherosclerotic lesion.
The precise mechanisms by which lipids induce the lesions, and the identity of the lipidic atherogenic agent(s) is not yet clear. Epidemiologic and clinical studies have established that the incidence of atherosclerosis is positively related to serum cholesterol levels (Epstein, F. H., In "International conference on Atherosclerosis", Carlson, L. A., et al., eds, Raven Press, New York (1978)), with the risk of disease correlating more strongly with an increase in the low-density lipoprotein fraction (LDLC) and/or a decrease in the high-density fraction (HDLC) (Wallace, R. B. and Anderson, R. A., Epidemiol Rev 9:95 (1987)). Results from the Framingham Study suggest that the ratio of LDLC/HDLC is the best predictor in. men closely followed by TC/HDLC (Kannel, W. B., Am Cardiol 52:9B (1983)). However, a number of other lipidic risk factors have also been identified such as beta-very low density lipoprotein (beta-VLDL) (Mahley, R. W., et al., J. Lip. Res. 21:970 (1980)), triglycerides (TG) in intermediate-density lipoprotein (IDL) in women (Dahlen, G. H., et al., Circulation 74:758-65 (1986)), Lp (a) (Reardon, M. F., Circulation 71:881-8 (1985)) and apolipoproteins A-1 and B (Wallace R. B. and Anderson, R. A., Epidemiol. Rev. 9:95 (1987)).
PHB is an amphiphillic lipid which is well-known as a high molecular weight storage polymer in bacteria, in which it accumulates in cytoplasmic granules (MW range of 60,000 to 1,000,000) (Dawes, E. A., et al., Adv. Microb. Physiol. 10:135 (1973)). PHB is synthesized from acetyl-CoA by two major pathways--a three step synthesis as e.g. in Alcaligenes eutrophos or Zoologea ramigera (Schubert, P., et al., J. Bacteriol. 170:5837 (1988)) or a five step synthesis as in Rhodospirillum rubrum (Moskowitz, G. J., et al., Biochemistry 8: 2748 (1969)). The schematic diagram FIG. 1 shows the relationship between PHB and cholesterol syntheses. The first step in both biochemical pathways is the condensation of acetyl-CoA to acetoacetyl-CoA catalyzed by beta-ketothiolase. In the case of PHB synthesis, this step is followed by reduction with NADPH or NADH, whereas cholesterol synthesis requires that a third acetyl-CoA condense with acetoacetyl-CoA before the reductive step (Packter, N. M., In "Biosynthesis of Acetate-derived Compounds". John Wiley, New York pp 145-150 (1973)). Sharing the common intermediate-acetoacetyl-CoA-causes both PHB and cholesterol synthesis to be regulated by changes in intracellular concentrations of acetyl-CoA.
The work leading up to the discovery of PHB in the serum lipoproteins is summarized here. It has been established that a low molecular weight species of PHB (MW ca 15,000) exists in the plasma membranes of bacteria. This is complexed with calcium polyphosphate of approximately the same molecular size (Reusch, R. N., et al., J. Bacteriol 156:778 (1983); Reusch, R. N., et al., J. Bacteriol. 168:553 (1986); Reusch, R. N., et al., Can. J. Microbiol. 33:435 (1987); Reusch, R. N., et al., Proc. Natl. Acad. Sci. USA 85:4176 (1988)). The location, composition and putative structure of this complex suggest it may be involved in Ca.sup.2+ and PO.sub.4.sup.2- transport, and consequently may play a role in calcium regulation and signaling. The potential importance of the PHB membrane complex coupled with the ubiquitous distribution of the PHB monomer, R-beta-hydroxybutyrate, prompted us to search for PHB and the PHB complex in eukaryotes. We surveyed a variety of plant and animal tissues and found that PHB and its complex were widely distributed in biological cells (Reusch, R. N., Soc. Exp. Biol. Med. 191:377 (1989)). The intracellular location of PHB and its complexes in beef liver cells was primarily in the membrane fractions, particularly in mitochondria and microsomes with lesser but significant amounts in plasma membranes.